L-serine compositions, methods and uses for treating neurodegenerative diseases and disorders

ABSTRACT

L-serine, L-serine precursors, L-serine derivatives and L-serine conjugates for treatment, amelioration and/or prevention of protein aggregation/tangles/plaques and diseases associated with protein aggregation/tangles/plaques. In particular, treatments and uses for L-serine, L-serine precursors, L-serine derivatives and L-serine conjugates include Alzheimer&#39;s disease (AD), Parkinson&#39;s disease, Amyotrophic Lateral Sclerosis (ALS), and Huntington disease (HD).

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/683,821, filed Nov. 21, 2012, which claims priority to U.S.Application No. 61/562,194, filed Nov. 21, 2011, all of whichapplications are expressly incorporated herein by reference in theirentirety.

INTRODUCTION

Protein translation is a highly efficient and accurate process forassembling the 20 standard amino acids into proteins. Error rates intranslation are relatively rare (1 in 10³ to 10⁴) and rely on theability of the system to discriminate between the 20 protein (orcanonical) amino acids (Zaher, et al., Cell 136, 746-762 (2009)).However such rare errors which result in the mischarging of tRNAsynthetase by the wrong amino acid can result in misfolded or truncatedproteins, and subsequent cell damage, such as in the sti (sticky)mutation in mice where alanine is substituted for glutamic acid,resulting in neurodegeneration (Lee, et al., Nature 443, 50-55 (2006)).We here report that a nonprotein amino acid produced by cyanobacteria,β-methylamino-L-alanine (BMAA), can be mistakenly incorporated intohuman proteins. We also report that this misincorporation can beinhibited by L-serine.

Protein translation is the process whereby the genetic code isinterpreted from information contained in the nucleic acid sequence inmRNA into the primary sequence of a polypeptide chain. Fidelity ofprotein synthesis, at the translational level, relies on the specificityof amino acid and cognate tRNA recognition by tRNA synthetases. Incertain cases, when two protein amino acids have a similar structure, aproofreading step checks the “fit” of an amino acid to the catalyticsite of the tRNA synthetase and the bond is hydrolysed if the wrongamino acid is bound (Zaher, et al., Cell 136, 746-762 (2009)). Hundredsof non-protein amino acids exist; many occur naturally in plants, someare produced in vivo from amino acid oxidation, others are syntheticallyproduced (Rubenstein, et al. Medicine (Baltimore) 79, 80-89 (2000);Rodgers, et al., Free Radic Biol Med 32, 766-775 (2002); Rodgers, etal., Int J Biochem Cell Biol 40, 1452-1466 (2008); Bell, J Agric FoodChem 51, 2854-2865 (2003)). Nonprotein amino acids which are closestructural analogues of any of the 20 protein amino acids can bind to anamino acid-tRNA synthetase and become mistakenly peptide bonded into apolypeptide chain (Rodgers, et al., Int J Biochem Cell Biol 40,1452-1466 (2008)). In order to be incorporated into proteins,non-protein amino acids have to be present in the cell at sufficientlevels to successfully compete with the protein amino acid for chargingonto tRNA. Examples of non-protein amino acids which are misincorporatedinto proteins causing pathological effects include cavananine and L-DOPA(Rubenstein, et al. Medicine (Baltimore) 79, 80-89 (2000); Rodgers, etal., Int J Biochem Cell Biol 40, 1452-1466 (2008); Allende, et al., JBiol Chem 239, 1102-1106 (1964); Rosenthal, et al., Science 196, 658-660(1977); Rosenthal Quarterly Review of Biology, 52, 155-178 (1977)).

BMAA has been found to be associated with proteins in cyanobacteria andother organisms (Banack, et al., Mar Drugs 5, 180-196 (2007); Cox, etal., Proc Natl Acad Sci USA 102, 5074-5078 (2005); Murch, et al., ProcNatl Acad Sci USA 101, 12228-12231 (2004)). The possibility that thecyanobacterial toxin BMAA is misincorporated into proteins was bolsteredby a study in which autoradiographic analysis was performed on miceafter a single injection of ³H-BMAA reported ‘a distribution patternsimilar to that of a protein-forming amino acid’ (Karlsson, et al.,Pigment Cell Melanoma Res 22, 120-130 (2009)). ³H-BMAA uptake wasdemonstrated in tissues with high levels of protein synthesis, andradioactivity was maintained in the tissue after acid extraction(Karlsson, et al., Pigment Cell Melanoma Res 22, 120-130 (2009)). Thesedata are consistent with incorporation of BMAA by protein synthesis.

As disclosed herein, a nonprotein amino acid produced by cyanobacteria,β-methylamino-L-alanine (BMAA), can be mistakenly incorporated intohuman proteins. The misincorporation of BMAA can be inhibited byL-serine.

SUMMARY

As disclosed herein, L-Serine can block the insertion of BMAA in humancell cultures, preventing protein misfolding. Significantly, human cellsthat are at risk of undergoing programmed cell death via apoptosis canbe rescued with the addition of L-Serine.

Alzheimer's Disease (AD) is a progressive neurodegenerative disease thatimpairs cognition and memory. The cause of this most common form ofdementia is not known. There are familial and sporadic forms ofAlzheimer's and known risk factors that affect the disease.

One of the hallmarks of Alzheimer's pathology is aggregated amyloid betapeptide (Aβ), along with other ubiquitinated and cytoskeletalproteinaceous materials. Aβ (predominantly 40 or 42 amino acids inlength) is derived from the proteolytic cleavage of the full-lengthamyloid precursor protein (APP). APP is a membrane protein that containsCu(II) and Zn(II) metal-ion binding sites in the tail portion thatcontains the Aβ region. Though Aβ containing plaques are part of thepathology of the disease, very little is known about the function ofboth this smaller peptide and the full-length APP. The plaque inducing42-amino acid long Aβ protein is produced from the normal cleavage ofAPP into the various subforms. There are two pathways for APP shown inthe figure at right, indicating the amyloidogenic path (left) to Aβ,sAPPβ and C99 or the non-amyloidogenic path (right) to p3, sAPPβ, andC83.

Accordingly, L-serine, as well as L-serine precursors, L-serinederivatives and L-serine conjugates, is a drug candidate for thetreatment, amelioration and/or prevention of proteinaggregation/tangles/plaques and diseases associated with proteinaggregation/tangles/plaques such as Alzheimer's disease (AD),Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), Huntington'sdisease (HD) and as well as a treatment that can slow or stop diseaseprogression or continuing neurodegeneration.

In accordance with the invention, there are provided methods and usesfor L-serine, or a precursor, derivative or conjugate of L-serine in:preventing, inhibiting or reducing incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into one or more proteins of a mammalian cell; and preventing,inhibiting or reducing misfolding or aggregation of one or more proteinsin a mammalian cell. In one embodiment, a method or use includescontacting a cell with L-serine, or a precursor, derivative or conjugateof L-serine, in an amount sufficient to prevent, inhibit or reduceincorporation of β-N-methylamino-L-alanine (BMAA), or a derivative orisomer of BMAA, into the protein. In another embodiment, a method or useincludes contacting the cell with L-serine, or a precursor, derivativeor conjugate of L-serine, in an amount sufficient to prevent, inhibit orreduce misfolding or aggregation of the protein.

In accordance with the invention, there are also provided methods anduses for L-serine, or a precursor, derivative or conjugate of L-serinein: reducing or decreasing risk of a neurological disease or disordercaused or characterized by misfolding or aggregation of one or moreproteins in a subject; stabilizing, or reducing or inhibitingprogression of, a neurological disease or disorder caused orcharacterized by misfolding or aggregation of one or more proteins in asubject; and treating a neurological disease or disorder caused orcharacterized by misfolding or aggregation of one or more proteins in asubject. In one embodiment, a method or use includes administering tothe subject L-serine, or a precursor, derivative or conjugate ofL-serine, in an amount sufficient to reduce or decrease risk of theneurological disease or disorder caused or characterized by misfoldingor aggregation of one or more proteins. In another embodiment, a methodor use includes administering to the subject L-serine, or a precursor,derivative or conjugate of L-serine, in an amount sufficient tostabilize, or reduce or inhibit progression of, a neurological diseaseor disorder a caused or characterized by misfolding or aggregation ofone or more proteins. In a further embodiment, a method or use includesadministering to the subject L-serine, or a precursor, derivative orconjugate of L-serine, in an amount sufficient to treat the neurologicaldisease or disorder caused or characterized by misfolding or aggregationof one or more proteins.

Cells include mammalian, such as primate (e.g., human) cells. Particularnon-limiting examples of cells include a neuron or a glial cell.Particular non-limiting examples of proteins include a TAR DNA-bindingprotein 43 (TDP-43) and alpha-synuclein.

Precursors, derivatives and conjugates of L-serine include an L-serinepolymer (polyserine), a salt of L-serine, an alkylated L-serine or anL-serine lipid. Precursors, derivatives and conjugates of L-serine alsoinclude salts, such as a sodium salt, a potassium salt, a calcium salt,a magnesium salt, a zinc salt, or an ammonium salt of L-serine.Precursors, derivatives and conjugates of L-serine further include analkylated L-serine, such as L-serine with an alkyl group, e.g., an alkylcomprising 1-20 carbon atoms. Precursors, derivatives and conjugates ofL-serine moreover include an L-serine ester, an L-serine di-ester, aphosphate ester of L-serine, a sulfate or sulfonate ester of L-serine, apegylated L-serine, a lipidated L-serine or an L-serine with one or morepolyethylene glycol (PEG) moieties. A non-limiting example of aprecursor of L-serine is L-phosphoserine.

Precursors, derivatives and conjugates of L-serine can be formulatedinto a composition or formulation, such as a pharmaceutical compositionor formulation. Precursors, derivatives and conjugates of L-serine canalso be included in liposomes or micelles. Such compositions andformulations, including pharmaceutical compositions, formulations,liposomes and micelles, include those suitable for administration ordelivery by any route, such as orally, by injection, by infusion, byintubation, via catheter, intraspinally, or intracranially.

Derivatives and isomers of BMAA include, but are not limited to,2,4-diaminobutyric acid (2,4-DAB), 2,3-diaminobutyric acid (2,3-DAB),N-(2-aminoethyl)glycine (AEG), or β-amino-N-methyl-alanine (BAMA).

Neurological diseases and disorders include diseases and disorderscharacterized by protein misfolding or protein aggregates, for example,histologically. Neurological diseases and disorders include diseases anddisorders also include those characterized by motor or cognitivedeficiency. Specific clinical forms of neurological diseases anddisorders treatable in accordance with the methods and uses hereininclude Alzheimer's disease, Parkinson's disease, Amyotrophic LateralSclerosis (ALS), Progressive Supranuclear Palsy (PSP), Lewy BodyDementia (LBD), Amyotrophic Lateral Sclerosis/Parkinsonism DementiaComplex (ALS/PDC), Huntington's disease (HD), Pick's disease andFrontotemporal Dementia (FTD).

Non-limiting examples of a symptom of a neurological disease or disorderinclude, for example, motor or cognitive deficiency; fatigue; tremors;ataxia; slurred, thick or irregular speech; muscle cramps, twitching,atrophy or weakness; shortness of breath; eating, breathing orswallowing difficulty; short term or long term memory loss; difficultyconcentrating or completing familiar or routine tasks; space and timeconfusion; vision, color or sign recognition loss; depth perceptionloss, speaking or writing difficulty; loss of judgment; vocabulary loss;moodiness; irritability; aggression; paranoia; delusions; withdrawalfrom social engagement; stiffness or rigidity; loss of fine or grossmotor control; slowing of movement; impaired balance; body instability;posture or gait abnormality; shuffling walk; reduced coordination;physical instability; unsteady gait; motor dysfunction; jerky bodymovement; slowed saccadic eye movement; body rigidity; seizure;difficulty chewing, eating, swallowing or speaking; deterioration incognition/mental capabilities; dementia; sleep, behavioral orpsychiatric abnormalities; difficulty in speech or thinking; behavioralchanges; impaired regulation of social conduct; passivity; lethargy;social withdrawal; inertia; over-activity; pacing; wandering; loss ofbalance; lunging forward when mobilizing; fast walking; imbalance (e.g.,bumping into objects or people); falls; changes in personality; slowingof movement; loss of inhibition or ability to organize information;slurred speech; difficult swallowing; opthalmoparesis or impaired eyemovement; impaired eyelid function; facial muscle contracture; Neckdystonia or backward tilt of the head with stiffening of neck muscles;sleep disruption; urinary/bowel incontinence; or parkinsonism.

Methods and uses of the invention include those that provide asubjective or objective improvement in any symptom of a neurologicaldisease or disorder, as set forth herein, or that is known to one ofskill in the art. In particular embodiments, a method or use prevents,reduces or inhibits onset, severity, frequency, or duration of one ormore symptoms of a neurological disease or disorder.

Methods and uses of the invention include administration, delivery orcontact of a subject, or any tissue organ or cell of a subject, with anycompatible means for delivery or contact of L-serine, precursor,derivative or conjugate of L-serine. In particular embodiments,L-serine, precursor, derivative or conjugate of L-serine, isadministered orally, by injection, by infusion, by intubation, viacatheter or intracranially to a subject in a method or use. In moreparticular aspects, L-serine, precursor, derivative or conjugate ofL-serine, is administered at least once daily for at least one week to asubject; at least two, three, four, five, six, seven, 8, 9, 10, 11 or 12weeks to a subject or at least one, two, three, four, five, six, seven,8, 9, 10, 11 or 12 months to a subject.

Methods and uses of the invention include doses of L-serine, precursor,derivative or conjugate of L-serine, optionally intended to achieve adesired effect. In particular embodiments, L-serine, precursor,derivative or conjugate of L-serine, is administered at a dose of about1-10 mg/day, 10-25 mg/day, 25-50 mg/day, 50-100 mg/day, 100-250 mg/day,250-500 mg/day, 500-750 mg/day, 750-1,000 mg/day, 1,000-2,000 mg/day,2,000-3,000 mg/day, 3,000-4,000 mg/day, 4,000-5,000 mg/day, 5,000-7,500mg/day, 7,500-10,000 mg/day, 10-15 g/day, 15-20 g/day, 20-25 g/day,25-30 g/day, 30-40 g/day, 40-50 g/day 50-75 g/day or 75-100 g/day to asubject; is administered at a dose of about 1-10 mg/kg body weight,10-25 mg/kg body weight, 25-50 mg/kg body weight, 50-100 mg/kg bodyweight, 100-250 mg/kg body weight, 250-500 mg/kg body weight, 500-750mg/kg body weight, or 750-1,000 mg/kg body weight to a subject; or isadministered at a dose of about 1-10 mg/kg body weight per day, 10-25mg/kg body weight per day, 25-50 mg/kg body weight per day, 50-100 mg/kgbody weight per day, 100-250 mg/kg body weight per day, 250-500 mg/kgbody weight per day, 500-750 mg/kg body weight per day, or 750-1,000mg/kg body weight per day to a subject.

The invention also provides methods for identifying an agent thatreduces, inhibits or prevents incorporation of β-N-methylamino-L-alanine(BMAA), or a derivative or isomer of BMAA, into a protein. In oneembodiment, a method includes contacting a serine racemase with a testcompound in the presence of L-serine under conditions where the L-serinewould be converted to D-serine by the serine racemase; and determiningif the test compound inhibits or reduces conversion of the L-serine toD-serine by the serine racemase. If the test compound inhibits orreduces conversion of the L-serine to D-serine by the serine racemasethe test compound is identified as an agent that reduces or inhibits orprevents incorporation of β-N-methylamino-L-alanine (BMAA), or aderivative or isomer of BMAA, into a protein.

The invention further provides methods for identifying a candidate agentfor reducing or inhibiting or preventing incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into a protein. In one embodiment, a method includes contacting a serineracemase with L-serine under conditions where the L-serine would beconverted to D-serine by the serine racemase in the presence of a testagent; and determining if the test agent inhibits or reduces conversionof the L-serine to D-serine by the serine racemase. An inhibition orreduction identifies the test agent as a candidate agent for reducing orinhibiting or preventing incorporation of β-N-methylamino-L-alanine(BMAA), or a derivative or isomer of BMAA, into a protein.

The invention moreover provides methods of screening for an agent thatreduces or inhibits or prevents incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into a protein. In one embodiment, a method includes contacting a serineracemase with L-serine under conditions where the L-serine would beconverted to D-serine by the serine racemase in the presence a testagent; and determining if the test agent inhibits or reduces conversionof the L-serine to D-serine by the serine racemase, thereby screeningfor an agent that reduces or inhibits or prevents incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into a protein.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1B show uptake and incorporation of ³H-BMAA into proteins byMRC-5 cells. MRC-5 cells were incubated with ³H-BMAA (31.25 nM) in HBSScontaining 10% FCS. After 2, 4, and 16 hours, cells were lysed and theprotein concentration and level of radioactivity in the lysate and cellproteins determined. 1A: Uptake of radiolabel by cells was expressed asdisintegrations per minute (DPM) per μg of cell protein. 1B: Radiolabelin the cell protein fraction was expressed as DPM per μg of total cellprotein

FIG. 2 shows inhibition of incorporation of radiolabel into cell proteinby cycloheximide (CHX). MRC-5, HUVEC and SH-SY5Y cells were incubatedwith ³H-BMAA (31.25 nM) with or without CHX 2 μg/ml for 16 hours. Cellsproteins were isolated and the amount of radiolabel present in cellproteins expressed as DPM per cell protein. The amount of radiolabelpresent in cell proteins in the CHX-treated cultures was expressedrelative to that of control cultures (no CHX) which was set at 100%.Parallel cultures of MRC-5 cells were incubated with ³H-leucine (41 nM)with or without CHX 2 μg/ml [MRC-5 (Leu)] and effects of CHX similarlydetermined (open bars). *** indicates P<0.001 using Student's two-tailedt-test.

FIG. 3 shows removal of radiolabel from cell proteins followingincubation of cells with ³H-BMAA. SH-SY5Y cells were incubated with³H-BMAA (31.25 nM) for 24 hours. Cell proteins were isolated by TCA (5%)precipitation. The amount of radiolabel released from proteins (ie. notTCA precipitable) after incubation at 37° C. with DTT (1 mM) and SDS(2%) with DTT (DTT/SDS) are shown relative to that of control samplesincubated with buffer alone. Cell proteins were also incubated withpronase (2 mg/ml) for 48 hours at 37° C. or HCl (12 M) for 12 hours andthe release of radiolabel quantified relative that of buffer alone (forpronase) or water (for HCl). All protein samples were processed intriplicate.

FIGS. 4A-4C show inhibition of incorporation or radiolabel into cellproteins by L-serine. 4A: MRC-5 cells were incubated with ³H-BMAA (31.25nM) for 16 hours in the presence of L-serine (0, 50, 100 and 250 μM).Cell proteins were isolated by TCA precipitation and radiolabel in cellproteins expressed as disintegrations per minute (DPM) per μg cellprotein. Inhibition of incorporation of radiolabel at each L-serineconcentration is shown relative to cells cultured in medium containingno L-serine. 4B: MRC-5 cells were incubated with 3H-BMAA (31.25 nM) for16 hours in the presence of 250 μM L-Serine (L-SER) or 250 μM D-serine(D-SER) and the incorporation of radiolabel compared to cells cultureswith no L-serine (CTR, set at 100%). 4C: MRC-5 cells were incubated with³H-BMAA (31.25 nM) for 16 hours in the presence of all 20 protein aminoacids at 400 μM (NONE), or Hank's buffered salt solution containing noamino acids (ALL) or in presence of all 19 protein amino acids withL-serine omitted (L-SERINE) and the radiolabel in cell proteinsexpressed as disintegrations per second (DPM).

FIG. 5 shows protein-bound BMAA in MRC5 cells incubated with free-BMAAin the culture medium. Peak normalized by concentrations of lysine.

FIGS. 6A-6D show incubation with BMAA results in the formation ofautofluorescent bodies and apoptotic changes in cells. Autofluorescencewas observed in MRC-5 cells incubated with BMAA (300 μM) for 96 hours(6A) and this was prevented by co-incubation with L-serine (300 μM)(6B). Fluorescent microscopy of MRC-5 cells dual-stained of withacridine orange to detect apoptosis and ethidium bromide to detectnecrosis, revealed morphological changes in BMAA-treated cell and theappearance of “pale” cells indicative of cells undergoing apoptosis (6D)which were absent in cells that were not exposed to BMAA (6C). There wasa significant increase in Annexin V staining in SH-SY5Y cells incubatedwith 500 μM BMAA alone showed as measured using flow cytometry.Co-incubation with CHX (2 μg/mL) or L-serine (50 μM) significantlyreduced Annexin V binding to cells, indicative of a reduction inapoptosis (6E).

FIG. 7 shows that L-serine reduces BMAA incorporation into proteins.

FIG. 8 shows that L-serine promotes Drosophila survival and blocks BMAAinduced mortality.

FIG. 9 shows that BMAA impairs zebrafish swimming performance.

FIGS. 10A-10C show that BMAA injection causes abnormalities in neuronaldevelopment. 10A: growth and branching of motor neurons was clearlyobserved after 30 hours. 10B and 10C: In eggs injected with L-BMAA,truncated neurons (indicated by white arrows) were observed.

DETAILED DESCRIPTION

As disclosed herein, BMAA can be incorporated into proteins leading toundesirable protein misfolding, protein aggregation and consequentneuronal disorders and diseases characterized by proteinaggregation/tangles/plaques. L-serine can inhibit or prevent BMAAincorporation into proteins thereby decreasing or preventing orundesirable protein aggregation and consequent neuronal disorders anddiseases characterized by protein aggregation/tangles/plaques.Accordingly, L-serine, as well as L-serine precursors, L-serinederivatives and L-serine conjugates, can be used as a treatment forneuronal disorders and diseases characterized by proteinaggregation/tangles/plaques.

In accordance with the invention, there are provided methods and usesfor preventing, inhibiting or reducing incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into one or more proteins (e.g., present in a cell), and methods anduses for preventing, inhibiting or reducing misfolding or aggregation ofone or more proteins of a cell (e.g., mammalian cell). In oneembodiment, a method or use includes contacting a cell with L-serine, ora precursor, derivative or conjugate of L-serine, in an amountsufficient to prevent, inhibit or reduce incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAAwhich mischarges serine aminoacyl-tRNA synthetase, into a protein of thecell (e.g., mammalian cell). Isomers of BMAA include 2,4-diaminobutyricacid (2,4-DAB), 2,3-diaminobutyric acid (2,3-DAB),N-(2-aminoethyl)glycine (AEG), and β-amino-N-methyl-alanine (BAMA)(Banack et al, Toxicon 56, 868-879 (2010); Banack et al, Toxicon 57,730-738 (2011); Jiang et al, Anal Bioanal Chem 403, 1719-1730 (2012)).In another embodiment, a method or use includes contacting the cell withL-serine, or a precursor, derivative or conjugate of L-serine, in anamount sufficient to prevent, inhibit or reduce misfolding oraggregation of the protein of the cell (e.g., mammalian cell).

In accordance with the invention, there are also provided methods anduses for reducing or decreasing risk of a neurological disease ordisorder caused or characterized by misfolding or aggregation of one ormore proteins in a subject. In one embodiment, a method or use includesadministering to the subject L-serine, or a precursor, derivative orconjugate of L-serine, in an amount sufficient to reduce or decreaserisk of the neurological disease or disorder caused or characterized bymisfolding or aggregation of one or more proteins.

In accordance with the invention, there are further provided methods anduses of stabilizing, preventing or reducing or inhibiting progressionof, a neurological disease or disorder caused or characterized bymisfolding or aggregation of one or more proteins in a subject. In oneembodiment, a method or use includes administering to the subjectL-serine, or a precursor, derivative or conjugate of L-serine, in anamount sufficient to stabilize, or reduce or inhibit progression of, aneurological disease or disorder a caused or characterized by misfoldingor aggregation of one or more proteins.

In accordance with the invention, there are additionally providedmethods and uses of treating a neurological disease or disorder causedor characterized by misfolding or aggregation of one or more proteins ina subject. In one embodiment, a method or use includes administering tothe subject L-serine, or a precursor, derivative or conjugate ofL-serine, in an amount sufficient to treat the neurological disease ordisorder caused or characterized by misfolding or aggregation of one ormore proteins.

Neurological disorders and diseases are characterized by abnormal ordeficient nerve or neuron cell numbers, function or activity. Such nerveand neuron cells may be present in the central nervous system (CNS,e.g., brain, spinal cord) or peripheral nervous system (PNS, outside thebrain and spinal cord, somatic, autonomic and sensory nervous system).Types of neurons affected by such disorders and diseases includeunipolar, bipolar and multipolar (e.g., motor) neurons.

L-serine, as well as L-serine precursors, L-serine derivatives andL-serine conjugates, can provide a detectable or measurable therapeuticbenefit or improvement to a subject. A therapeutic benefit orimprovement is any measurable or detectable, objective or subjective,transient, temporary, or longer-term benefit to the subject orimprovement in the disorder or disease, an adverse symptom, consequenceor underlying cause, of any degree, in a tissue, organ, cell or cellpopulation of the subject. Therapeutic benefits and improvementsinclude, but are not limited to, reducing or decreasing occurrence,frequency, severity, progression, or duration of one or more symptoms orcomplications associated with a disorder or disease, or an underlyingcause or consequential effect of the disorder or disease. L-serine, aswell as L-serine precursors, L-serine derivatives and L-serineconjugates, methods and uses therefore include providing a therapeuticbenefit or improvement to a subject.

Methods and uses of the invention include treatment methods and usesthat desirably result in an improvement in a symptom or underlying causeof the subject's disease or disorder, that is a change consideredbeneficial to the subject. Thus, treatment can result in an improvement,such as inhibiting, reducing or preventing a progression or worsening ofthe disease or disorder or symptoms, or further deterioration or onsetof one or more additional symptoms of the disease or disorder. Thus, asuccessful treatment outcome leads to a “therapeutic effect,” orinhibiting, reducing or preventing the severity or frequency of one ormore symptoms or underlying causes of a disease or disorder in thesubject.

The term “ameliorate” means a detectable or measurable objective orsubjective improvement in a subject's condition. A detectable ormeasurable improvement includes a subjective or objective reduction inthe severity or frequency of one or more symptoms caused by orassociated with the disorder or disease, or an improvement in theunderlying causes of the disorder or disease, or a reversal of thedisorder or disease.

Stabilizing a disease or disorder is also a successful treatmentoutcome. A successful treatment can reduce or prevent the severity orfrequency of one or more symptoms of the disease or disorder, inhibitprogression or worsening of the disease or disorder, and in someinstances, reverse the disease or disorder. Accordingly, in the case ofa neurological disease or disorder, for example, treatment can lead toan improvement of one or more symptoms of the neurological disease ordisorder, stabilizing one or more symptoms of the neurological diseaseor disorder, or a reversal of the neurological disease or disorder.

Non-limiting symptoms of neurological disorders and diseases include,but are not limited to: motor (e.g., coordination) or cognitivedeficiency; fatigue; tremors; ataxia; slurred, thick or irregularspeech; muscle cramps, twitching, atrophy or weakness (e.g., hands,arms, legs, swallowing, breathing, speech muscles); shortness of breath;eating, breathing or swallowing difficulty. Non-limiting symptoms ofneurological disorders and diseases also include, but are not limitedto: short term or long term memory loss; difficulty concentrating orcompleting familiar or routine tasks; space and time confusion; vision,color or sign recognition loss; depth perception loss, speaking orwriting difficulty; loss of judgment; vocabulary loss; moodiness;irritability; aggression; paranoia; delusions; and withdrawal fromsocial engagement. Non-limiting symptoms of neurological disorders anddiseases further include, but are not limited to: tremor; stiffness orrigidity; loss of fine or gross motor control; slowing of movement;impaired balance; body instability; posture or gait abnormality; andshuffling walk. Non-limiting symptoms of neurological disorders anddiseases additionally include, but are not limited to: reducedcoordination; physical instability; unsteady gait; motor dysfunction;jerky body movement (chorea); slowed saccadic eye movement; bodyrigidity; seizure; difficulty chewing, eating, swallowing or speaking;deterioration in cognition/mental capabilities including dementia; shortand/or long term memory loss; sleep, behavioral and psychiatricabnormalities (e.g., anxiety, depression, loss of emotional affect,aggression, compulsory behavior). Non-limiting symptoms of neurologicaldisorders and diseases moreover include, but are not limited to:difficulty in speech and thinking; behavioral changes; impairedregulation of social conduct (e.g. breaches of etiquette, tactlessness,dis-inhibition, criminal behavior); passivity; lethargy; socialwithdrawal; inertia; over-activity; pacing; wandering. Non-limitingsymptoms of neurological disorders and diseases still further include,but are not limited to: loss of balance; lunging forward whenmobilizing; fast walking; imbalance, e.g., bumping into objects orpeople; falls; changes in personality; slowing of movement; dementia(including loss of inhibition and ability to organize information);slurred speech; difficult swallowing; opthalmoparesis or impaired eyemovement (particularly in the vertical direction which accounts for someof the falls experienced by patients); impaired eyelid function; facialmuscle contracture; Neck dystonia or backward tilt of the head withstiffening of neck muscles; sleep disruption; urinary/bowelincontinence; and parkinsonism. Methods and uses, such as treatment inaccordance with the invention, include treatment that improves orameliorates any one of the foregoing symptoms, to any degree or durationof time.

Methods and uses, such as treatment in accordance with the invention,also include affecting the underlying causes of the disease or disorderthereof. Thus, in the case of a neurological disease or disorder, forexample, stabilizing or decreasing worsening of the condition, forexample, by objective and subjective measures of clinical severity ofthe neurological disorder is considered a successful treatment outcome.For example, the Amyotrophic Lateral Sclerosis Functional Rating Scale(ALSFRS) is a 10-item functional inventory devised for use intherapeutic Amyotrophic Lateral Sclerosis (ALS) trials. Functions suchas eating, grooming, ambulation (motility and motor skills),communication and others are part of the scale. Accordingly, animprovement in the underlying cause of ALS can be reflected by animprovement in one or more of the functional inventories, for example.

In a method or use of the invention in which a therapeutic benefit orimprovement is a desired outcome, L-serine, or a precursor, derivativeor conjugate of L-serine, can be administered in a sufficient oreffective amount to a subject in need thereof. An “amount sufficient” or“amount effective” refers to an amount that is calculated or likely toprovide, in single or multiple doses, alone or in combination with oneor more other compositions (therapeutic agents such as achemotherapeutic or immune stimulating drug), treatments, or therapeuticprotocols, regimens or agents, a detectable response of any duration oftime (long or short term), a desired outcome in or a benefit to asubject of any measurable or detectable degree or for any duration oftime (e.g., for hours, days, months, years, or cured). The dose or“sufficient amount” or “effective amount” for treatment (e.g., toprovide a therapeutic benefit or improvement) typically is effective toameliorate a disorder or disease, or one, multiple or all adversesymptoms, consequences or complications of the disorder or disease, to ameasurable extent, although reducing or inhibiting a progression orworsening (e.g., stabilizing) of the disorder or disease or a symptom,is considered a satisfactory outcome.

Treatment can therefore result in inhibiting, reducing or preventing adisorder or disease, or an associated symptom or consequence, orunderlying cause; inhibiting, reducing or preventing a progression orworsening of a disorder or disease, symptom or consequence, orunderlying cause; or further deterioration or occurrence of one or moreadditional symptoms of the disorder or disease, or symptom. Thus, asuccessful treatment outcome leads to a “therapeutic effect,” or“benefit” or inhibiting, reducing or preventing the occurrence,frequency, severity, progression, or duration of one or more symptoms orunderlying causes or consequences of a disorder or disease, in thesubject. Treatment methods and uses affecting one or more underlyingcauses of the disorder or disease or a symptom are therefore consideredbeneficial. Stabilizing or inhibiting or reducing progression orworsening of a disorder or disease is also a successful treatmentoutcome.

A therapeutic benefit or improvement therefore need not be completeablation of any one, most or all symptoms, complications, consequencesor underlying causes associated with the disorder or disease. Thus, asatisfactory endpoint is achieved even where only an incrementalimprovement in a subject's condition is achieved, such as a partialreduction in the occurrence, frequency, severity, progression, orduration, or inhibition or reversal, of one or more associated adversesymptoms or complications or consequences or underlying causes,worsening or progression (e.g., stabilizing one or more symptoms orcomplications of the disease or disorder), of one or more of thephysiological, biochemical or cellular manifestations or characteristicsof the disorder or disease, over a short or long duration of time(minutes, hours, days, weeks, months, etc.).

A therapeutic benefit or treatment efficacy can be observed or measuredby improvement in any one or more of the symptoms that characterize orare associated with the neurological disorder or disease a set forthherein. Particular non-limiting examples of therapeutic benefit orimprovement for a neurological disorder or disease include, but is notlimited to any of the following symptoms (e.g., a reduction or decreaseof an adverse symptom or progression of an adverse symptom): motor(e.g., coordination) or cognitive deficiency; fatigue; tremors; ataxia;slurred, thick or irregular speech; muscle cramps, twitching, atrophy orweakness (e.g., hands, arms, legs, swallowing, breathing, speechmuscles); shortness of breath; eating, breathing or swallowingdifficulty; short term or long term memory loss; difficultyconcentrating or completing familiar or routine tasks; space and timeconfusion; vision, color or sign recognition loss; depth perceptionloss, speaking or writing difficulty; loss of judgment; vocabulary loss;moodiness; irritability; aggression; paranoia; delusions; withdrawalfrom social engagement; tremor; stiffness or rigidity; loss of fine orgross motor control; slowing of movement; impaired balance; bodyinstability; posture or gait abnormality; shuffling walk; reducedcoordination; physical instability; unsteady gait; motor dysfunction;jerky body movement (chorea); slowed saccadic eye movement; bodyrigidity; seizure; difficulty chewing, eating, swallowing or speaking;deterioration in cognition/mental capabilities including dementia; shortand/or long term memory loss; sleep, behavioral and psychiatricabnormalities (e.g., anxiety, depression, loss of emotional affect,aggression, compulsory behavior); difficulty in speech and thinking;behavioral changes; impaired regulation of social conduct (e.g. breachesof etiquette, tactlessness, dis-inhibition, criminal behavior);passivity; lethargy; social withdrawal; inertia; over-activity; pacing;wandering; loss of balance; lunging forward when mobilizing; fastwalking; imbalance, e.g., bumping into objects or people; falls; changesin personality; slowing of movement; dementia (including loss ofinhibition and ability to organize information); slurred speech;difficult swallowing; opthalmoparesis or impaired eye movement(particularly in the vertical direction which accounts for some of thefalls experienced by patients); impaired eyelid function; facial musclecontracture; neck dystonia or backward tilt of the head with stiffeningof neck muscles; sleep disruption; urinary/bowel incontinence; andparkinsonism. Methods and uses, such as treatment in accordance with theinvention, include treatment that improves or ameliorates any one of theforegoing symptoms, to any extent or period of time.

Additional examples include measurement of various functions compared toestablished criteria. For example, an assessment of intellectual(cognitive) functioning such as memory testing or physical functioningcan further characterize state of a disease. A therapeutic benefit ortreatment efficacy of a neurological disorder or disease (or severity ofa neurological disorder or disease) can also be ascertained or measuredby mechanical means or instrumentation. In particular, positron electrontomography (PET) scan of the brain of a person with a neurologicaldisorder or disease can show the degree to which there is a loss offunction. Imaging such as computed tomography (CT) or magnetic resonanceimaging (MRI), or with single positron emission computed tomography(SPECT) or positron emission tomography (PET) can also be used toidentify cerebral pathology or subtypes of dementia in order toascertain therapeutic benefit or treatment efficacy of a neurologicaldisorder or disease, or severity of a neurological disorder or disease.

For Alzheimer's Disease (AD), a commonly used measure of cognitiveimpairment is the established NINCDS-ADRDA Alzheimer's Criteria fordiagnosis. Eight cognitive domains are most commonly impaired in AD,namely, memory, language, perception (visual color, depth, symbolrecognition), attention constructive/functional abilities, orientationand problem solving abilities. These domains can therefore be useful toascertain diagnosis and as such any improvement of AD. A PET scan of thebrain of a person with Alzheimer's disease classically shows biparietalhypometabolism in the temporal lobe.

For Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig'sdisease), diagnosis is primarily based on the symptoms and signs, andtests to rule out other diseases. A neurologic examination is used toassess whether symptoms such as muscle weakness, atrophy of muscles,hyper-reflexia, and spasticity are present, and over time, becomeprogressively worse. One of these tests that distinguishes ALS fromother diseases or disorders having similar symptoms is electromyography(EMG), a recording technique that detects electrical activity inmuscles. Certain EMG findings can support the diagnosis of ALS. Anothercommon test measures nerve conduction velocity (NCV). Specificabnormalities in NCV may suggest, for example, that the patient has aform of peripheral neuropathy (damage to peripheral nerves) or myopathy(muscle disease) rather than ALS. MRI scans are often normal in patientswith ALS, but it can reveal evidence of other problems that may becausing the symptoms, such as a spinal cord tumor, a herniated disk inthe neck, syringomyelia, or cervical spondylosis.

For Parkinson's Disease, diagnosis of onset can be made following theappearance of physical and/or psychological symptoms specific to thedisease, as set for the herein, for example. Characteristic featuresinclude tremor; body stiffness or rigidity; loss of fine or gross motorcontrol; slowing of movement; impaired balance; body instability;posture or gait abnormality; and shuffling walk, reduced coordination;physical instability; unsteady gait; motor dysfunction; jerky bodymovement (chorea); slowed saccadic eye movement; seizure; difficultychewing, eating, swallowing or speaking; deterioration incognition/mental capabilities including dementia; short and/or long termmemory loss; sleep, and behavioral and psychiatric abnormalities (e.g.,anxiety, depression, loss of emotional affect, aggression, compulsorybehavior).

For Huntington's disease (HD), diagnosis of onset can be made followingthe appearance of physical and/or psychological symptoms specific to thedisease, as set for the herein, for example. Excessive unintentionalmovements of any part of the body, which are abrupt and have randomtiming and distribution, suggest a diagnosis of HD. Cognitive orpsychiatric symptoms are rarely the first diagnosed; they are usuallyonly recognized in hindsight or when they develop further. Diseaseprogression can be measured using the unified Huntington's diseaserating scale which provides an overall rating based on motor,behavioral, cognitive, and functional assessments. Medical imaging, suchas CT and MRI, can show atrophy of the caudate nuclei early in thedisease, but these changes alone are not diagnostic of HD. Cerebralatrophy can be seen in the advanced stages of the disease. Genetictesting can be used to confirm a physical diagnosis if there is nofamily history of HD. Even before onset of symptoms, a genetic test canconfirm if an individual carries an expanded copy of the trinucleotideCAG repeats in the HTT gene that causes the disease. Because HD followsan autosomal dominant pattern of inheritance, individuals who are atrisk can be readily identified. A positive result can be obtained manyyears before symptoms begin. A negative test means that the individualdoes not carry the expanded copy of the gene and will not develop HD.

For Pick's Disease (PD), the most widely known criteria is from the UKParkinson's Disease Society Brain Bank and the U.S. National Instituteof Neurological Disorders and Stroke. The PD Society Brain Bank criteriarequires slowness of movement (bradykinesia) plus either rigidity,resting tremor, or postural instability, while ruling out other possiblecauses for these symptoms. In addition, three or more of the followingfeatures are required during onset or evolution: unilateral onset,tremor at rest, progression in time, asymmetry of motor symptoms,response to levodopa for at least five years, clinical course of atleast ten years and appearance of dyskenisias induced by the intake ofexcessive levodopa.

For Frontotemporal Dementia (FTD), diagnosis is primarily clinicalincluding changed behaviors, changes in language, combined withneuropsychological tests and imaging. Structural MRI scans often revealfrontal lobe and/or anterior temporal lobe atrophy, but in early casesthe scan may appear normal. Atrophy is often asymmetric. Comparison ofimages taken at different time points (e.g. a year apart) can showevidence of atrophy in two cross-sectional images that may be reportedas normal. FDG-PET scans classically show frontal and/or anteriortemporal hypometabolism, which also helps differentiate FTD fromAlzheimer's disease.

An amount sufficient or an amount effective can but need not be providedin a single administration and, can but need not be, administered aloneor in combination with another composition, treatment, protocol ortherapeutic regimen. For example, the amount may be proportionallyincreased as indicated by the need of the subject, status of thedisorder or disease treated or the side effects of treatment. Inaddition, an amount sufficient or an amount effective need not besufficient or effective if given in single or multiple doses without asecond composition, treatment, protocol or therapeutic regimen, sinceadditional doses, amounts or duration above and beyond such doses, oradditional compositions, treatments, protocols or therapeutic regimensmay be included in order to be considered effective or sufficient in agiven subject.

Amounts considered sufficient also include amounts that result in areduction of the use of another treatment, therapeutic regimen orprotocol. For example, a sufficient or effective amount of L-serine, ora precursor, derivative or conjugate of L-serine, is considered ashaving a therapeutic effect if administration results in less amount orfrequency of a drug, or another therapy or protocol, being required totreat a neurological disorder or disease.

An amount sufficient or an amount effective need not be effective ineach and every subject treated, prophylactically or therapeutically, ina particular subject, or a majority of treated subjects in a given groupor population. As is typical for treatment or therapeutic methods, somesubjects will exhibit greater or less response to a given treatment,therapeutic regimen or protocol. An amount sufficient or an amounteffective refers to sufficiency or effectiveness in a given subject, nota group or the general population. Such amounts will depend in part uponthe disease or disorder treated, such as the type or stage (early oradvanced) of the disorder or disease, the therapeutic effect desired, aswell as the individual subject (e.g., the bioavailability within thesubject, gender, age, etc.).

An amount sufficient for the methods, uses and compositions of theinvention include about 1-10 milligrams (mg), 10-25 mg, 25-50 mg, 50-100mg, 100-250 mg, 250-500 mg, 500-750 mg, 750-1,000 mg, 1,000-2,000 mg,2,000-3,000 mg, 3,000-4,000 mg, 4,000-5,000 mg, 5,000-7,500 mg,7,500-10,000 mg, 10-15 grams (g), 15-20 g, 20-25 g, 25-30 g, 30-40 g,40-50 g, 50-75 g or 75-100 g. Amounts sufficient can also be usedaccording to the mass of a subject (e.g., in Kilograms, kg). Forexample, for a human subject, amounts of L-serine, or a precursor,derivative or conjugate of L-serine, include about 1-10 mg/kg bodyweight, 10-25 mg/kg body weight, 25-50 mg/kg body weight, 50-100 mg/kgbody weight, 100-250 mg/kg body weight, 250-500 mg/kg body weight,500-750 mg/kg body weight, 750-1,000 mg/kg body weight, 1-5 g/kg bodyweight, or 5-10 g/kg body weight of a subject. Such amounts for themethods, uses and compositions of the invention can be less, forexample, from about 50-500, 500-5000, 5000-25,000 or 25,000-50,000ng/kg.

Methods and uses of the invention may be practiced prior to (i.e.prophylaxis) or after symptoms begin, before or after symptoms or thedisease or disorder develop. Administering L-serine, or a precursor,derivative or conjugate of L-serine, prior to or immediately followingdevelopment of a symptom may decrease the severity or frequency ofsymptoms, or the underlying cause of the neurological disorder, in thesubject. In addition, administering L-serine, or a precursor, derivativeor conjugate of L-serine, prior to or immediately following developmentof one or more symptoms may stabilize or slow progression or worseningof a symptom, or the underlying cause of the neurological disorder ordisease.

Methods and compositions of the invention may be used in vitro, ex vivoor in vivo. Compositions can be administered or delivered as a single ormultiple dosage form, on consecutive or alternating days orintermittently, to a subject. For example, single or multiple dosageforms can be administered or delivered on alternating days orintermittently, over about 1 to 7, or 7 to 45, or 45 to 90 days or overabout 1-4, 4-8, 8-12, 12-18, 18-24, 24-48, or more weeks, to a subject.

The term “contacting” means direct or indirect binding or interactionbetween two or more entities (e.g., between L-serine, a precursor,derivative or conjugate of L-serine, or molecule target, within a cell,for example). Contacting as used herein includes in solution, in solidphase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo canbe referred to as administering, or administration, or in vivo delivery.

The term “subject” refers to animals, typically mammalian animals, suchas a non-human primate (vervets, gorillas, chimpanzees, orangutans,macaques, gibbons), a domestic animal (dogs and cats), a farm animal(horses, cows, goats, sheep, pigs), experimental animal (mouse, rat,rabbit, guinea pig) and humans. Human subjects include adults andchildren. Human subjects include those having or at risk of having aneurological disorder. At risk subjects can be identified throughgenetic screening for predisposition towards a neurological disorder ora family history of a neurological disorder, for example, byaccumulation of BMAA in keratinaceous tissues or blood plasma (see,e.g., U.S. Pat. Nos. 7,256,002 and 7,670,783). Subjects further includedisease model animals (e.g., such as mice and non-human primates) fortesting in vivo efficacy of L-serine, or a precursor, derivative orconjugate of L-serine.

The invention also provides compositions, including L-serine, or aprecursor, derivative or conjugate of L-serine, in an amount that isable to produce one or more of the activities associated with L-serine.In one embodiment, a composition includes L-serine, or a precursor,derivative or conjugate of L-serine, in an amount sufficient to treat aneurological disorder. In another embodiment, a composition includes anamount of L-serine, or a precursor, derivative or conjugate of L-serine,sufficient to inhibit, reverse, ameliorate or reduce one or moresymptoms of a neurological disorder. In yet another embodiment, acomposition includes an amount of L-serine, or a precursor, derivativeor conjugate of L-serine, sufficient to reverse an underlying causes ofa neurological disorder.

Amounts of L-serine, or a precursor, derivative or conjugate ofL-serine, for the methods, uses and compositions of the inventioninclude about 1-10 milligrams (mg), 10-25 mg, 25-50 mg, 50-100 mg,100-250 mg, 250-500 mg, 500-750 mg, 750-1,000 mg, 1,000-2,000 mg,2,000-3,000 mg, 3,000-4,000 mg, 4,000-5,000 mg, 5,000-7,500 mg,7,500-10,000 mg, 10-15 grams (g), 15-20 g, 20-25 g, 25-30 g, 30-40 g,40-50 g, 50-75 g or 75-100 g. Amounts can also be produced and/orprovided according to the mass of a subject (e.g., in Kilograms, kg).For example, for a human subject, amounts of L-serine, or a precursor,derivative or conjugate of L-serine can be adjusted according to themass of the human. Such amounts of L-serine, or a precursor, derivativeor conjugate of L-serine, include about 1-10 mg/kg body weight, 10-25mg/kg body weight, 25-50 mg/kg body weight, 50-100 mg/kg body weight,100-250 mg/kg body weight, 250-500 mg/kg body weight, 500-750 mg/kg bodyweight, 750-1,000 mg/kg body weight, 1-5 g/kg body weight, or 5-10 g/kgbody weight of a subject. Such amounts can be less, for example, fromabout 50-500, 500-5000, 5000-25,000 or 25,000-50,000 ng/kg.

Methods and uses of the invention can be practiced using L-serine, or aprecursor, derivative or conjugate of L-serine, optionally in suchamounts as set forth herein. A derivative or conjugate of L-serine canbe a modification (e.g., a protecting group) at any one, any two or allthree functional groups, namely, the amino moiety (NH₂), acid moiety(—COOH) and/or the hydroxyl moiety (—OH) groups) of L-serine. Themodified groups can be cleaved in vivo to yield free L-serine and ahydrolyzed protecting group. Suitable groups are pharmaceuticallyacceptable and typically substantially non-toxic. A derivative orconjugate of L-serine may have enhanced stability and/or solubilitycompared to free L-serine which aids in storage and dosing, and uptakeof the L-serine active compound. These and other L-serine derivativesand conjugates known to one of skill in the art are included in theinvention methods, uses and compositions (e.g., pharmaceuticalformulations).

Non-limiting examples of L-serine derivatives and conjugates in which ahydroxyl moiety is derivatized to form a protected hydroxyl includeester, carbonate, phosphate, and sulfonate ester. The hydroxyl group ofL-serine can be selectively esterified with a suitable carbonyl,phosphonyl, or sulfonyl electrophile after prior protection of the aminoand acid moieties of the L-serine. Typically, one or both of the aminoand acid protecting groups can be removed after selective esterificationof the hydroxyl moiety; bio-compatibility of the acid and aminoprotecting groups is not an issue where both protecting groups areremoved. Non-limiting examples of amino protecting groups include butare not limited to tert-butoxycarbonyl (Boc) and carbobenzyloxy (CBz),which are removable under acidic and hydrogenolysis conditions,respectively. Suitable acid protecting groups include tert-butyl (tBu)and benzyl (Bn) esters, which are removable under acidic andhydrogenolysis conditions, respectively. Suitable hydroxyl protectiongroups, are esters of carboxylic acids, phosphoric acid, phosphonicacids, sulfuric and sulfonic acids and other hydroxyl protecting groupsknown to the skilled artisan. One of ordinary skill in the art willappreciate that hydroxyl-protected L-serine may exist as a zwitterionicspecies as in the free amino acid or may be readily converted to aprotic acid addition salt.

Non-limiting examples of L-serine derivatives and conjugates in whichthe amino moiety is derivatized include formation of an amide or urea,or carbamate-containing L-serine. The amino moiety of L-serine can beselectively derivatized after prior protection of the hydroxyl and acidmoieties of the L-serine. Both hydroxyl and acid moieties in L-serinecan be protected with groups amenable to removal after the finalderivatization of the amino group. Typically, one or both of thehydroxyl and acid moiety protecting groups are removed after selectivederivatization of the L serine amino moiety.

L-serine derivatives and conjugates include derivatization of the acidmoiety, which can be selectively derivatized after protecting thehydroxyl and amino moieties of the L-serine. Both hydroxyl and aminomoieties in L-serine can be masked with protecting groups that areamenable to removal after the final protection of the acid moiety.Typically, one or both of the hydroxyl and amino moiety protectinggroups are removed after selective derivatization of the L-serine acidmoiety.

In various additional embodiments, an L-serine conjugate includes apolymer. For example, L-serine can be within a small (e.g., 2-10)residue peptide which includes other amino acids. In this embodiment,enzymes such as peptidases and pepsin can cleave the peptide intoindividual amino acids, releasing L-serine. In such embodiments, thepeptide can include two, three, four, five, six, seven, eight, nine,ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., or more L-serinemolecules Ser-[Ser]n where n is 1 to 1000. The administered peptideyields serine or smaller serine peptides which are in turned hydrolyzed.

Starting materials useful for preparing L-serine derivatives andconjugates are commercially available or can be prepared by well-knownsynthetic methods (Harrison et al., “Compendium of Synthetic OrganicMethods”, Vols. 1-8 (John Wiley and Sons, 1971-1996); “BeilsteinHandbook of Organic Chemistry,” Beilstein Institute of OrganicChemistry, Frankfurt, Germany; Feiser et al, “Reagents for OrganicSynthesis,” Volumes 1-17, Wiley Interscience; Trost et al,“Comprehensive Organic Synthesis,” Pergamon Press, 1991; “Theilheimer'sSynthetic Methods of Organic Chemistry,” Volumes 1-45, Karger, 1991;March, “Advanced Organic Chemistry,” Wiley Interscience, 1991; Larock“Comprehensive Organic Transformations,” VCH Publishers, 1989; Paquette,“Encyclopedia of Reagents for Organic Synthesis,” John Wiley & Sons,1995). Other methods for synthesis of hydroxyl-protected serine compoundand amino acid protection groups will be readily apparent to the skilledartisan.

L-serine, and precursors, derivatives and conjugates of L-serine, andcompositions thereof (e.g., pharmaceutical formulations), may beadministered systemically, regionally or locally by any route. Forexample, L-serine, or a precursor, derivative or conjugate of L-serine,may be administered intravenously, orally (e.g., ingestion),intracranially, intraspinally, intramuscularly, intraperitoneally,intradermally, subcutaneously, intracavity, transdermally (topical),parenterally, e.g. transmucosal and rectally. Methods, uses and serine,and precursors, derivatives and conjugates of L-serine, and compositionsthereof, of the invention including pharmaceutical formulations can beadministered via a microencapsulated delivery system or packaged into animplant for sustained, continuous or intermittent administration.

Compositions further include pharmaceutical formulations containingL-serine, or a precursor, derivative or conjugate of L-serine. Suchpharmaceutical formulations can be formulated in an amount having one ormore of the activities disclosed herein, and a pharmaceuticallyacceptable carrier or excipient. In various embodiments, apharmaceutical formulation includes L-serine, or a precursor, derivativeor conjugate of L-serine in an amount sufficient to achieve an intendedeffect.

As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, excipients, diluents andthe like that can be administered to a subject, preferably withoutproducing excessive adverse side-effects (e.g., nausea, abdominal pain,headaches, etc.). Such preparations for administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions.

Pharmaceutical formulations can be made from carriers, diluents,excipients, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with administration to a subject. Such formulations canbe contained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir. Supplementary active compounds and preservatives, among otheradditives, may also be present, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.

A pharmaceutical formulation can be formulated to be compatible with itsintended route of administration. Thus, pharmaceutical formulationsinclude carriers, diluents, or excipients suitable for administration byroutes including intraperitoneal, intradermal, subcutaneous, oral (e.g.,ingestion or inhalation), intravenous, intracavity, intracranial,intraspinal, transdermal (topical), parenteral, e.g. transmucosal andrectal.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following: a sterile diluentsuch as water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical formulations suitable for injection include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. Fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride can beincluded in the composition. Prolonged absorption of injectableformulations can be achieved by including an agent that delaysabsorption, for example, aluminum monostearate or gelatin.

For oral administration, L-serine, or a precursor, derivative orconjugate of L-serine, or composition thereof, can be incorporated withexcipients in the form of tablets, troches, or capsules, e.g., gelatincapsules. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included in oral formulations. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidani such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or flavoring.

Formulations can also include carriers to protect L-serine, or aprecursor, derivative or conjugate of L-serine against degradation orelimination from the body, such as a controlled release formulation,including materials that slowly degrade within the body and in turnrelease the active ingredient(s). For example, a time delay materialsuch as glyceryl monostearate or glyceryl stearate alone, or incombination with a wax, may be employed.

Additional formulations include biodegradable or biocompatible particlesor polymeric substances such as polyesters, polyamine acids, hydrogel,polyvinyl pyrrolidone, polyanhydrides, polyglycolic acid,ethylene-vinylacetate, methylcellulose, carboxymethylcellulose,protamine sulfate, or lactide/glycolide copolymers,polylactide/glycolide copolymers, or ethylenevinylacetate copolymers inorder to control delivery of an administered composition. Methods forpreparation of such formulations are known to those skilled in the art.The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc., for example.

The rate of release of L-serine, or a precursor, derivative or conjugateof L-serine can be controlled by altering the concentration orcomposition of such macromolecules. For example, L-serine, or aprecursor, derivative or conjugate of L-serine can be entrapped inmicrocapsules prepared by coacervation techniques or by interfacialpolymerization, for example, by the use of hydroxymethylcellulose orgelatin-microcapsules or poly (methylmethacrolate) microcapsules,respectively, or in a colloid drug delivery system. Colloidal dispersionsystems include macromolecule complexes, nano-capsules, microspheres,microbeads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. These can be prepared accordingto methods known the skilled artisan, for example, as described in U.S.Pat. No. 4,522,811.

Additional pharmaceutical formulations appropriate for administrationare known in the art and are applicable in the methods, uses andcompositions of the invention (see, e.g., Remington's PharmaceuticalSciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The MerckIndex (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; andPharmaceutical Principles of Solid Dosage Forms, Technonic PublishingCo., Inc., Lancaster, Pa., (1993)).

L-serine, or a precursor, derivative or conjugate of L-serine of theinvention can include combinations of other compositions, and beincluded in the pharmaceutical compositions of the invention. Forexample, a drug that is used to treat a neurological disorder can beincluded with L-serine, or a precursor, derivative or conjugate ofL-serine.

L-serine, or a precursor, derivative or conjugate of L-serine, includingpharmaceutical formulations thereof can be packaged into kits, whichoptionally can contain instructions for use, for example, practicing amethod or use of the invention. The invention therefore provides kits.In one embodiment, a kit includes L-serine, or a precursor, derivativeor conjugate of L-serine, and/or a pharmaceutical formulation, packagedinto suitable packaging material. In additional embodiments, a kitincludes a label or packaging insert for practicing a method of theinvention. Thus, in one embodiment, a kit includes instructions fortreating a subject having or at risk of having a neurological disorder,in vitro, in vivo, or ex vivo. In additional embodiments, a kit includesa label or packaging insert including instructions for treating asubject having a neurological disorder in vivo, or ex vivo.

As used herein, the term “packaging material” refers to a physicalstructure housing the components of the kit. The packaging material canmaintain the components sterilely, and can be made of material commonlyused for such purposes (e.g., paper, corrugated fiber, glass, plastic,foil, ampules, etc.). The label or packaging insert can includeappropriate written instructions, for example, practicing a method oruse of the invention. Kits of the invention therefore can additionallyinclude instructions for using the kit components in a method or use ofthe invention.

Instructions can include instructions for practicing any of the methodsor uses of the invention described herein. Thus, L-serine, or aprecursor, derivative or conjugate of L-serine and pharmaceuticalcompositions thereof can be included in a container, pack, or dispensertogether with instructions for administration to a subject. Instructionsmay additionally include indications, a satisfactory clinical endpoint,any adverse symptoms that may occur, or additional information requiredby the Food and Drug Administration for use on a human subject.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin the kit, on a label affixed to the kit or packaging material, orattached to a vial or tube containing a component of the kit.Instructions may comprise voice or video tape which can optionally beincluded on a computer readable medium, such as a disk (hard disk),optical CD such as CD- or DVD-ROM/RAM, magnetic tape, electrical storagemedia such as RAM and ROM and hybrids of these such as magnetic/opticalstorage media.

Invention kits can also include one or more drugs that provide asynergistic or additive effect or that reduce or ameliorate one or moresymptoms of a neurological disorder. For example, a drug that reduces ordecreases a symptom of a neurological disorder may be included.Invention kits can additionally include a buffering agent, apreservative, or a stabilizing agent. The kit can further includecontrol components for assaying an activity or effect of treatment. Eachcomponent of the kit can be enclosed within a separate individualcontainer. For example, a kit can include a single unit dose ofL-serine, or a precursor, derivative or conjugate of L-serine as setforth herein. Alternatively, a kit can include multiple unit doses ofL-serine, or a precursor, derivative or conjugate of L-serine. Forexample, each of the multiple unit doses would contain an amount ofL-serine, or precursor, derivative or conjugate of L-serine, in aseparate individual container. Kit components can be in a mixture of oneor more containers and all of the various containers can be withinsingle or multiple packages.

The invention provides cell-free and cell-based methods of screeningfor, detecting and identifying agents that modulate serine racemaseactivity, and methods of screening, detecting and identifying agentsthat modulate incorporation of β-N-methylamino-L-alanine (BMAA), or aderivative or isomer of BMAA into a protein. The methods can beperformed in solution, in solid phase, in silica, in vitro, in a cell,and in vivo.

In one embodiment, a method of screening for an agent includescontacting serine racemase under conditions allowing L-serine to beconverted into D-serine in the presence a of test agent; and determiningif the test agent inhibits or reduces conversion of L-serine intoD-serine. In another embodiment, a method of identifying an agent thatreduces or inhibits or prevents incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into a protein includes contacting a serine racemase with a testcompound in the presence of a L-serine under conditions where theL-serine is converted to D-serine by the serine racemase and determiningif the test compound inhibits or reduces conversion of the L-serine toD-serine by the serine racemase. A reduction or inhibition of serineracemase converting L-serine to D-serine in the presence of the testagent or test compound identifies the test agent or test compound as anagent that decreases, reduces or inhibits or reduces conversion ofL-serine into D-serine, or that reduces or inhibits or preventsincorporation of β-N-methylamino-L-alanine (BMAA), or a derivative orisomer of BMAA, into a protein.

In a further embodiment, a method of identifying a candidate agent forreducing or inhibiting or preventing incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into a protein, includes contacting a serine racemase with L-serineunder conditions where the L-serine would be converted to D-serine bythe serine racemase in the presence of a test agent; and determining ifthe test agent inhibits or reduces conversion of the L-serine toD-serine by the serine racemase. An inhibition or reduction of D-serineidentifies the test agent as a candidate agent for reducing orinhibiting or preventing incorporation of β-N-methylamino-L-alanine(BMAA), or a derivative or isomer of BMAA, into a protein.

In an additional embodiment, a method of screening for an agent thatreduces or inhibits or prevents incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into a protein, includes contacting a serine racemase with L-serineunder conditions where the L-serine would be converted to D-serine bythe serine racemase in the presence a test agent; and determining if thetest agent inhibits or reduces conversion of the L-serine to D-serine bythe serine racemase. The foregoing method thereby screens for an agentthat reduces or inhibits or prevents incorporation ofβ-N-methylamino-L-alanine (BMAA), or a derivative or isomer of BMAA,into a protein.

Such methods can include further or additional method steps. Forexample, methods of identifying, detecting and screening can includealso measuring the activity of the agent to reduce or inhibit or preventincorporation of β-N-methylamino-L-alanine (BMAA), or a derivative orisomer of BMAA into a protein in a cell.

The terms “determining,” “assaying” and “measuring” and grammaticalvariations thereof are used interchangeably herein and refer to eitherqualitative or quantitative determinations, or both qualitative andquantitative determinations. When the terms are used in reference tomeasurement or detection, any means of assessing the relative amount,including the various methods set forth herein and known in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All patents, applications, publications, other references, GenBankcitations and ATCC citations cited herein are expressly incorporated byreference herein in their entirety. In case of conflict, thespecification, including definitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “L-serine, or a precursor, derivative or conjugateof L-serine” includes a plurality of L-serine, or precursors,derivatives or conjugates of L-serine, and so forth.

As used herein, all numerical values or numerical ranges includeintegers within such ranges and fractions of the values or the integerswithin ranges unless the context clearly indicates otherwise. Thus, forexample, reference to a range of 1-10, includes 1, 2, 3, 4, 5, etc., aswell as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc.,and so forth. A reference to a range includes a reference to subrangeswithin that range. Thus, for example, reference to 1-10 also include1-3, 1-4, 1-5, 1-6, 2-4, 2-5, 2-6, 2-7, 3-5, 3-6, 3-7, 3-8, etc.Reference to a series of ranges, for example, reference to a range of1-10 mg, 10-25 mg, 25-50 mg, 50-100 mg, 100-250 mg, 250-500 mg, 500-750mg, 750-1,000 mg, 1,000-2,000 mg, 2,000-3,000 mg, 3,000-4,000 mg,4,000-5,000 mg, 5,000-7,500 mg, 7,500-10,000 mg, 10-15 g, 15-20 g, 20-25g, 25-30 g, 30-40 g, 40-50 g, 50-75 g and 75-100 g include combinationsof combined ranges, such as 10-50, 50-500, 70-100 mg or g, etc. A seriesof ranges include both lower and upper ends of those ranges combinedinto ranges. Thus, for example, reference to a series of ranges such as50-100, 100-200, and 200-300, includes a range of 50-200, 50-300,100-300, etc.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include aspects that are not expressly included inthe invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1

This example describes various materials and methods.

Materials & Methods

MRC-5 cells were from American Tissue and Cell Culture (Virginia, USA).SH-SY5Y cells were from the European Collection of Cell Culture (ECACC).³H-BMAA (80 Ci/mmol, 0.5 mCi/mL) was obtained from American RadiolabeledChemicals. Dulbecco's modification of Eagle's minimum essential medium(DMEM) and HAMS F12 were from JRH biosciences, (Lenexa, Kansans, USA).BMAA, dithiothreitol, L-serine, D-serine, acridine orange, ethidiumbromide, cycloheximide and SDS were from Sigma Chemical Co.(Sigma-Aldrich, Castle Hill, NSW, Australia). BCA protein reagent wasfrom Pierce Biotechnology (Rockford, Ill., USA). BD Pharminigen™ AnnexinV-FITC apoptosis detection kit was from BD Biosciences (SydneyAustralia). Water was from a Milli Q 4 stage system (Millipore-Waters,Lane Cove, NSW, Australia). All HPLC equipment was supplied by theShimadzu Corporation (Kyoto, Japan) except the column (Nova-Pak® C18 4μM 3.9×300 mm) and the AccQ•Tag derivitization kit which were suppliedby Waters Corporation (MA, USA). Other chemicals, solvents andchromatographic materials were AR or HPLC grade.

Cell Culture

MRC-5 cells, a human lung fibroblast cell line (passage number 14-19),and SH-SY5Y cells, a human neuroblastoma cell-line (passage number30-32), were maintained in DMEM, or DMEM/Hams F12 respectivelycontaining 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/mLpenicillin, and 100 μg/mL streptomycin at 37° C. in a humidifiedatmosphere of 5% CO₂ and 95% air. Human umbilical vein endothelial cells(HUVEC) were harvested enzymatically with type II collagenase(Sigma-Aldrich) under sterile conditions as described by Minter (Minter,et al., Thromb Haemost 67, 718-723 (1992)) and established as primarycell cultures in M199 (Trace Biosciences, Sydney, Australia) containing20% FBS, 4 mM L-glutamine, 0.5% endothelial cell growth promoter, 100U/mL penicillin, and 100 μg/mL streptomycin. All media were preparedwith endotoxin-free water (Baxter) and filtered with Zetapore filters(Cuno Life Sciences Division). Cells were seeded at 3×10⁵ cells per wellin 6 well plates and allowed to adhere overnight (16 hours) beforetreatment.

Studies Examining Incorporation of BMAA into Proteins and Inhibition ofIncorporation by Cycloheximide and Amino Acids

MRC-5 cells were incubated with ³H-BMAA (31.25 nM) in HBSS containing10% FCS. After 2, 4, and 16 hours, cells were washed three times inphosphate buffered saline and lysed by freeze-thawing in Triton X-100.The protein concentration in the lysate was determined using thebicinchoninic acid assay (BCA) (Smith, et al., Anal Biochem 150, 76-85(1985)) (and radiolabel in the cell lysate quantified by liquidscintillation counting (LSC). Cell proteins were then isolated bytrichloroacetic acid (TCA) (5%) precipitation washed three times in TCA(5%) and dissolved in formic acid.

To determine if incorporation of radiolabel into proteins was proteinsynthesis dependent MRC-5, SH-SY5Y and HUVEC cells were incubated with³H-BMAA (31.25 nM) with or without CHX 2 μg/ml for 16 hours. The amountof radiolabel present in cell proteins in the CHM-treated cultures wasexpressed relative to that of control cultures, which was set at 100%.Parallel cultures of MRC-5 cells were incubated with ³H-leucine (41 nM)with or without CHX 2 μg/ml under identical culture conditions andprocessed as before.

To examine the ability of individual amino acids to reduce incorporationor radiolabel into proteins, MRC-5 cells were incubated with ³H-BMAA(31.25 nM) for 16 hours in the presence of individual amino acids (250μM) and the radiolabel present in the cell proteins assessed as before.All of the 20 protein amino acids (L-isomers) were examined individuallyin triplicate cell cultures. To confirm that L-serine had an inhibitoryeffect on incorporation of radiolabel into proteins, MRC-5 cells wereincubated with ³H-BMAA (31.25 nM) for 16 hours in the presence ofL-serine (0, 50, 100 and 250 μM) and in a separate experiment with 250μM L-serine and D-serine. Incorporation of radiolabel was determinedrelative to cells incubated in medium containing no serine. MRC-5 cellswere then incubated with ³H-BMAA (31.25 nM) for 16 hours in HBSS alone,HBSS containing all 20 protein amino acids (400 μM) or 19 protein aminoacids except L-serine and the amount of radiolabel in cell proteinassessed as before.

Removal of Radiolabel from Cell Proteins Generated by Incubating MRC-5Cells with 3H-BMAA

SH-SY5Y cells were incubated with ³H-BMAA (31.25 nM) for 24 hours andcell proteins isolated by TCA (5%) precipitation. Proteins were washedthree times in TCA (5%), rinsed in ice-cold acetone and re-dissolved inPBS. The amount of radiolabel released from the proteins (ie. not TCAprecipitable) after incubation at 37° C. with DTT (1 mM) and SDS (2%)with DTT (DTT/SDS) was determined by Liquid Scintillation Counting(LSC). Cell proteins were also incubated with pronase (2 mg/mL) for 48hours in 100 μM Tris HCl buffer pH 8 containing 20 mM CaCl₂ at 37° C. orin HCl (12 M) for 12 hours and the release of radiolabel quantifiedrelative that of buffer alone (for pronase) or water (for HCl). Allprotein samples were processed in triplicate.

Recovery of Incorporated BMAA from Proteins Following Hydrolysis

After incubation with ³H-BMAA or cold BMAA, cells were washed threetimes with PBS, lysed by freeze thawing in Triton-X-100 (0.1%) and cellproteins precipitated in TCA (5%). Protein pellets were washed threetimes with TCA (5%) and hydrolysed in boiling 6 M HCl at 110° C. for 16hours as previously described (Mondo, et al., Mar Drugs 10, 509-520(2012)). Protein pellets were freeze-dried and reconstituted in 20 mMHCl. Particulates were removed by centrifugation through 0.22 μMmembranes and the hydrolysate derivatized in AccQ•Tag (WatersCorporation, Australia). Amino acids were separated on a Waters C-18column using a method and gradient as described previously (Mondo, etal., Mar Drugs 10, 509-520 (2012)). For recovery of radiolabel inhydrolysed samples, fractions were collected manually every minute for40 minutes, diluted into 5 ml scintillant (Ultima Gold™ scintillant,Perkin Elmer) and distintegrations per minute (DPM) determined by LSC.

Autofluorescence Imaging of Cells

MRC-5 cells were incubated in DMEM supplemented with 300 μM BMAA in thepresence or absence of 300 μM L-serine for 96 hours with daily mediumchanges. Cellular autofluorescence was visualized using an invertedfluorescent microscope (Olympus IX71) as described previously (Dunlop,et al., Biochem J 410, 131-140 (2008)).

Lactate Dehydrogenase (LDH) Assay

Lactate dehydrogenase release from cells was determined as describedpreviously (Tang, et al., Phytother Res 25, 417-423 (2010)).

Acridine Orange (AO)/Ethidium Bromide (EtBr) Dual-Staining of Cells

MRC-5 cells were incubated in the presence and absence of 500 μM BMAAfor 23 hours. After removing the medium, the cells were rinsed once withwarm PBS and incubated in a solution of AO/EB then visualised usingfluorescent microscopy as described previously (Tang, et al., PhytotherRes 25, 417-423 (2010)).

Binding of Annexin V to Phosphatidylserine (PS) Exposed on the PlasmaMembrane

Late-stage apoptosis or necrosis was measured by simultaneous stainingwith propidium iodide and Annexin V using the BD Pharminigen™ AnnexinV-FITC apoptosis detection kit and flow cytometry performed as describedpreviously (Dunlop, et al., Biochem J 435, 207-216 (2010)).

Extraction Methods for Fruit Fly BMAA Analysis

Thirty flies were from each treatment were weighed and sonicated (FisherScientific sonic dismembrator model 100; 2 watts for 30 secs) in 10%trichloroacetic acid (TCA 72 μg/μl). TCA extraction was completed in twosteps 20 h at 4° C. using one half the TCA volume followed bycentrifugation (13 rpm for 3 min, Labnet spectrafuge 16M) and removal ofsupernatant. This was followed by a second sonication and extraction, 5hours at room temperature using an equal volume of TCA, followed bycentrifugation and removal of supernatant. The supernatants were pooled(with the exception of 50 μl removed from the last extraction) andcentrifuge filtered (0.22 μm Ultrafree-MC, Millipore). The remainingprotein pellet was transferred to a glass vial and hydrolyzed in 6 M HClfor 16 hours at 110° C. (58 μg/μl). A portion of the pooled TCA extractwas also hydrolyzed in an equal volume of 12 M HCl for 16 hours at 110°C. Extracts were diluted and derivatized with6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and analyzed byLC-MS/MS.

Analytical Methods for Fruit Fly BMAA

6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivates (AQC WatersAccQTag reagent, PN WAT052880) were analyzed using a triple quadrupoleinstrument (Thermo Scientific Finnigan TSQ Quantum UltraAM, San Jose,Calif.) after separation by a Waters Acquity-UHPLC system with a BinarySolvent Manager, Sample Manager and a Waters AccQTag Ultra column (part#186003837, 2.1×100 mm) at 55° C. Separation was achieved using gradientelution at 0.65 ml/min in aqueous 0.1% (v/v) formic acid (Eluent A) and0.1% (v/v) formic acid in acetonitrile (Eluent B): 0.0 min=99.1% A; 0.5min=99.1% A curve 6; 2 min=95% A curve 6; 3 min=95% A curve 6; 5.5min=90% A curve 8; 6 min=15% A curve 6; 6.5 min=15% A curve 6; 6.6min=99.1% A curve 6; 8 min=99.1% A curve 6. This gradient providedseparation of BMAA (Irvine Chemistry, CA) from isomers2,4-diaminobutyric acid (Sigma #32830 St. Louis, Mo.) andN-2(amino)ethylglycine (TCI America (Portland, Oreg.). Nitrogen gas wassupplied to the heated electrospray ionization (H-ESI) probe with anebulizing pressure of 40 psi and a vaporizer temperature of 400° C.

The mass spectrometer was operated under the following conditions: thecapillary temperature was set at 270° C., capillary offset of 35, tubelens offset of 110, auxiliary gas pressure of 35, spray voltage 3500,source collision energy of 0, and multiplier voltage of −1585. Thesecond quadrupole was pressurized to 1.0 mTorr with 100% argon.Product-ion analysis of AQC derivatized BMAA used m/z 459 (ionized witha single charge) and m/z 230 (ionized with a double charge) as theprecursor ions for collision-induced dissociation (CID).

Two-step mass filtering was performed during selective reactionmonitoring (SRM) of BMAA after CID in the second quadrupole, monitoringthe following transitions: m/z 459 to 119, CE 21 eV; m/z 459 to 289 CE17 eV; m/z 459 to 171 CE 38 eV; m/z 459 to 258 CE 21 eV; m/z 230 to 171CE 21 eV. Product ions were detected within the third quadrupole andtheir relative abundances were quantified.

Identification of BMAA was confirmed by comparison with an authenticatedstandard (Irvine Chemistry, CA) based upon four parameters (a) thepresence of the parent ions m/z 459 and m/z 230; (b) retention time; (c)presence of product ions from collision-induced dissociation (transitionm/z 459 to 171 quantifier ion; transitions m/z 459 to 289, 258, and 119qualifier ions; and transition m/z 230 to 171 qualifier ion); (d) ratiosof qualifier ions relative to the quantifier ion from parent ion m/z459. The adequacy of the AQC reaction was monitored by the examinationof L-lysine (Sigma #L5501) comparing peak areas of single derivatizedlysine (m/z 317) with those of double derivatized lysine (m/z 487).

Example 2

This example describes data indicating that BMAA is incorporated intoproteins, which can lead to protein misfolding/aggregation, and whichincorporation and misfolding/aggregation is inhibited by L-serine.

Incubation of human MRC-5 fibroblasts with ³H-BMAA in culture mediumdepleted in amino acids resulted in a time-dependent increase inradiolabel in cell lysates (FIG. 1A), a proportion of which wasassociated with cell proteins (FIG. 1B). Co-incubation of MRC-5 cellswith ³H-BMAA along with the protein synthesis inhibitor CHXsignificantly reduced the amount of radiolabel in the protein fraction(FIG. 2). CHX inhibited incorporation of the protein amino acid³H-leucine into proteins to the same extent as ³H-BMAA (FIG. 2)suggesting that ³H-BMAA was incorporated into proteins by a proteinsynthesis-dependent mechanism. ³H-BMAA was also found to be proteinassociated after incubation with human primary endothelial cells (HUVEC)and human neuroblastoma cells (SH-SY5Y), this process was again found tobe protein synthesis dependent since it was inhibited by CHX (FIG. 2).

To further examine the association between BMAA and cell proteins,availability of a range of treatments to release radiolabel from cellproteins was analyzed. Radiolabelled cell proteins were generated byincubating SH-SY5Y cells with ³H-BMAA for 24 hours. The radiolabel couldnot be removed from the isolated cell proteins by incubation with a 100fold molar excess of the reducing agent dithiothreitol (DTT) or byheating with the detergent sodium dodecylsulphate (SDS) as well as DTT(FIG. 3). Release of radiolabel required cleavage of peptide bonds byeither acid hydrolysis or proteolytic digestion with pronase (FIG. 3).

To determine which amino acid was being replaced by BMAA competitionbetween all 20 protein amino acids and ³H-BMAA for incorporation intocell proteins was analyzed. Incorporation of ³H-BMAA into cell proteinswas inhibited in the presence of L-serine in a concentration-dependentmanner (FIG. 4A). D-serine, which is not charged by mammalian tRNAsynthetases, did not prevent incorporation of ³H-BMAA into protein (FIG.4B). Incubation of cells with ³H-BMAA in culture medium which containedall 20 protein amino acids (400 μM) greatly reduced the amount ofradiolabel in the protein fraction relative to amino acid-depletedculture conditions (FIG. 4C); when L-serine was omitted from the aminoacid mixture, incorporation of BMAA into proteins significantlyincreased (FIG. 4C).

To further confirm that BMAA was present in cell proteins andincorporation was inhibited by L-serine, MRC-5 cells were incubated witha range of concentrations of BMAA (250-1000 μM) or BMAA with L-serine(250-1000 μM) and hydrolysed cell proteins were analyzed by tandem massspectroscopy on a triple quadrupole LC/MS/MS. Retention times, uniquedaughter ions, and ratios of m/z transitions during collision-induceddissociation matched those of an authenticated BMAA standard (FIG. 5A).Incubation of cells with increasing concentrations of BMAA resulted inincreasing recovery of BMAA from the hydrolyzed proteins (FIG. 5B).Consistent with the studies utilizing ³H-BMAA, incorporation ofnon-labeled BMAA was inhibited by both L-serine and CHX (FIG. 5C). Incells incubated for 96 hours with BMAA (300 μM), autofluorescent bodieswere evident in the nucleus, cytosol and in the perinuclear space (FIG.6A). This is consistent with the accumulation of aggregated proteins,since fluorescent pigment has been shown to accumulate in cells as aresult of aging (lipofuscin) or from a range of pathologies associatedwith impaired proteolysis (ceroid).

Co-incubation with L-serine prevented the formation of autofluorescentbodies (FIG. 6B). MRC-5 and SH-SY5Y cells did not show signs of necrosisas evidenced by no significant changes in LDH release (data notshown?—it's not) however, apoptosis was present as indicated byincreased staining for Annexin V (FIG. 6E) and the increased number of“pale” appearance of cells treated with acridine orange and ethidiumbromide (FIGS. 6C and D). Apoptosis could be abrogated by co-incubationwith L-serine as well as by CHX, which provides further evidence thatthis process is dependent upon BMAA incorporation into the protein chain(FIG. 6E). Taken together these data suggest that incorporation of BMAAinto cell proteins is a protein synthesis-dependent process, which isinhibited by L-serine.

Based on the foregoing data, the ability of BMAA to be mistakenlyincorporated into proteins in place of L-serine would increasemisfolding of aggregate-prone proteins. Post-mitotic cells such asneurons would be most affected since, amongst other factors(Polymenidou, et al., J Exp Med 209, 889-893 (2012)), they are unable todistribute protein aggregates amongst daughter cells. By promotingmisfolding of a number of disease-specific proteins, BMAA could betherefore be a single trigger for the complex neurological disordersreported on Guam in which amyotrophic lateral sclerosis, Parkinson'sdisease and dementia-like symptoms were evident in individuals exposedto BMAA (Bradley, et al., Amyotroph Lateral Scler 10 Suppl 2, 7-20(2009)).

Example 3

This example describes data indicating that L-Serine rescues Drosophilamelanogaster (fruit flies) from BMAA induced mortality, and additionaluseful models.

APP is evolutionarily conserved from invertebrates to vertebrates. Thisallows research to be performed on APP in Drosophila fruit flies, tostudy human diseases such as Alzheimer's disease. The Drosophila modelwas used to characterize how BMAA influences the production of theAPP-derived fragments, especially the amyloidogenic fragments.

The Drosophila melanogaster (fruit fly) insect model is a convenientinvertebrate system to measure important features of a human disease,including plaque formation, memory loss, social interactions, and theoverall neuronal structure of a model organism. Fruit flies alsoengineered to produce extra human Aβ will be fed BMAA and the resultingincrease or decrease in plaque inducing Aβ will be measured. Flies arean excellent model because their genetic characteristics have beenwell-worked out and a whole host of human diseases are examined in fruitflies.

Preliminary studies show the ability of L-Serine to preventmisincorporation of BMAA into proteins. In a series of feedingexperiments, Drosophila fruit flies were fed four different types ofmedia: 1) standard fruit fly media with 0 mM BMAA (control), 2) mediaenriched with 25 mM L-Serine, 3) media with 25 mM BMAA added, and 4)media with both 25 mM BMAA and 25 mM L-Serine added for three days offeeding. Misincorporation of BMAA into proteins was then analyzed usingtriple quadrupole LC/MS/MS and Orbital Trap LC/MS/MS. Addition ofL-Serine to the media reduced by half the amount of BMAAmisincorporation (FIG. 7).

Equally interesting is the analysis of the protective value of L-Serineon Drosophila dosed with BMAA. Fruit flies fed with BMAA enriched media(third set of blocks) suffered a 40% mortality after three days, whileflies fed with Serine plus BMAA (fourth set of blocks) had no mortality(FIG. 8). These flies were rescued by L-Serine.

There are two additional models that will be used to study the effectsof BMAA as follows:

1. Human neuronal cell lines (NT2 cells). These human neurons arecultured in petri dishes and are robust enough to study for long periodsof time. These cells are the closest model to what may occur at theneuronal level in the human brain. The ex-vivo cells carry all thefeatures important for neuron to neuron communication, neurotransmitterglutamate function and a convenient system for measuring cell viabilityand APP-fragments using protein (Western blot) techniques.2. Highly proliferating cells, denoted CHO/APP cells. These cells,derived from hamsters, have extra amounts of APP which will enablemeasurement of smaller Aβ fragments. Often, protein techniques whilegood at detecting small fragments, are further enhanced by using cellslines that have been engineered to produce more of a particular protein,in this case, APP.

Example 4

This example describes data in a vertebrate model that illustratesneurotoxicity of BMAA and that BMAA causes abnormalities in neuronaldevelopment.

The zebrafish (Dario rerio) is a clinically relevant model for humanneurological disorders and diseases such as spinal muscular atrophy,ataxia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington'sdisease, Parkinson's disease, dementia and Alzheimer's disease (see,e.g., Kabashi et al., Trends Genet. 26:373 (2010); and Kabashi et al.,Bicohim. Biophys. Acta 1812:335 (2011)). Zebrafish was used to determineBMAA toxicity, and to assess functional changes in swimming performancein fish following a single exposure to BMAA.

BMAA was directly injected through the chorion into the yolk, allowingit to directly compete with L-serine for incorporation into newlysynthesised proteins in the developing fish. BMAA (100 mg/ml in water)or water were delivered by an automated Picospritzer pump set to deliver5 nL per injection into the yolk (˜110 μl) of zebrafish immediatelyafter being fertilized. Fish were left to develop for 30 hours instandard conditions.

Survival:

In the control group 91% of the fish survived. In the BMAA-treatedgroup, zebrafish viability was reduced to 74%.

Surviving fish were reared in an aquarium equipped with biologicalfiltration and temp control set at 28° C. for 3 months. BMAA-treated andcontrol fish from the same parents were reared in the same tanks, thenseparated based on fluorescent tag prior to swimming assessment. After 3months all healthy adult fish with no deformations or abnormalities wereexamined. Critical swimming speed was determined, defined as the maximumswimming velocity that can be sustained for a set period in a watertunnel. Water was pumped from as reservoir (EcoTech Marine MP10 VortechPropeller Pump with EcoSMART Driver) through 50 mm diameter tubing. Amesh screen located in the downstream end prevented fish from leavingthe test section. Fish were swum for 6 minutes at setting 4 on the pump,then speed incrementally increased to setting 6 and maintained untilfish failed.

Swimming Performance:

BMAA-treated fish showed a significant impairment in swimmingperformance compared to the control fish (Ctrl) based on the time tofail (seconds) (FIG. 9). There was a significant difference between thetwo groups (p<0.001)

Neuronal Development:

Neurons were observed under a fluorescent microscope. In controlinjected fish (FIG. 10A), growth and branching of motor neurons could beclearly observed after 30 hours. In eggs injected with L-BMAA, truncatedneurons were observed (FIG. 10B, indicated by white arrows). Thedifference in total motor neuron length was quantitated by measuring thefirst ten motor neuron projections marked before the end of the yolkextension (Paquet D et al., J. Clin. Invest. 119:1382 (2009)).Measurements were conducted in three different fish per treatment usingthe freehand line tool in ImageJ (http://rsb.info.nih.gov/ij/ Version1.46r). Data was collated in GraphPad Prism (version 6 for OSX) andstatistical significance calculated using a Student's T-test. Horizontallines represent means of neuron length within and between samples (FIG.10C). P<0.0001.

Example 5

This example describes analysis of L-Serine prevention of BMAAmisincorporation into rodent neuroproteins.

A series of studies were conducted which demonstrated that radio-labeledBMAA, both triated and 14-C labeled BMAA, rapidly cross the blood-brainbarrier in rats and are inserted into neuroproteins. Studies testing todetermine if L-Serine blocks the misincorporation of radio-tagged BMAAinto rodent brains are undertaken.

Example 6

This example describes a Vervet model for evaluating the protectiveeffect of L-Serine against progressive neurodegenerative disease.

Non-human primates are exceptional models for understanding humandisease, since these animals share a large portion of the human genomeand have significant homologies with human nervous systems. Africangreen monkeys, known as vervets, are known to carry the APOe gene anddevelop cerebral amyloid-beta (Aβ) plaques, making vervets a promisingmodel for Alzheimer's disease (AD). One vervet study and one study foundthat Aβ vaccinations reduced Aβ in the brain. A subsequent human phase Itrial showed that the Aβ vaccinations were safe for people and thisavenue is currently being investigated largely due to the positivetrials in vervets, indicating that vervets are an accepted model forhuman Alzheimer's.

A colony of vervets was studied for protein misfolding and proteinaggregates produced by oral administration of BMAA and the ability ofequivalent doses of L-Serine to prevent BMAA misincorporation intoproteins, as well as L-serine to prevent protein misfolding and proteinaggregates. Vervets (16 animals) were divided into four differentgroups. Four of the vervets were fed 651 mg of BMAA daily. A secondgroup of four vervets were fed 651 mg of BMAA plus 651 mg of L-Serineeach day. The third group was dosed with 651 mg of L-Serine daily, andthe fourth control group received placebos, in this case 651 mg of riceflour. These doses, which normalize to 210 mg/kg/day, are comparable tothe doses used for macaques that induced acute neurotoxicity in someanimals with profound movement and cognitive deficits in the dosedanimals.

Neuroproteins derived from the vervet tissues as well as blood plasmaand cerebral spinal fluid samples are being analyzed with orbital trapmass spectrometry and triple quadrupole mass spectrometry for evidenceof BMAA misincorporation. In addition, the neuroproteins are studied forevidence of misfolding and aggregation using stains and fluorescentmicroscopy as previously detailed, as well as gross neuropathologicalstudies of brain tissues for lesions or other neuroanatomicaladnormalities.

Example 7

This example describes studies to evaluate Alzheimer's brains forproteins that have BMAA misincorporated into the protein.

Differences in the abundance of the enzymes, particularly 3-PGDH, PSAT,and PSPH that make endogenous L-Serine within primate astrocytes in theneuronal system body will be examined in order to understand ifdeficiencies in neuronal L-Serine makes neuroproteins more vulnerable toBMAA-misfolding. The specific make up of these Alzheimer's-relatedproteins can be used to prove that BMAA is being misincorporated inplace of L-Serine in living primate nervous systems. At the conclusionof these in vivo studies, a determination if L-Serine preventsmisincorporation of BMAA into neuroproteins, and if L-Serine is able toprevent subsequent protein misfolding and aggregation.

Extracts from Alzheimer's brains to purify and analyze for proteins thathave had BMAA misincorporated into the protein sequence will beperformed. Samples of brains from patients who suffered from Alzheimer'sdisease are obtained and proteins from the brains extracted. Extractswill be separated by electrophoresis on gels and the proteinstransferred to membranes. These membranes containing all the proteins inthe extracts will be probed with BMAA antibodies to determine whichproteins show reactivity with the antibodies and hence, which proteinscontain BMAA. The membrane probing will allow a calculation of the sizeor molecular weight of these proteins. An isolation of the proteins thathave the same molecular weight as for the proteins that were positivewhen assessed with membranes. These proteins will then be cut out fromthe gel, digested and analyzed using a mass spectrometer. The sequencesobtained for these digested proteins will be assessed using proteinanalysis software to identify the proteins based on their sequences andto also determine the sites where the mis-incorporation of BMAA occurs.

Alternatively, BMAA antibodies will be used to purify proteins fromsolutions by preparing antibody affinity columns. Protein extracts fromAlzheimer's brains will be subject to affinity purification. Afterpassing the protein solution through the antibody column, proteinscontaining BMAA will be more likely to bind to the antibodies on thecolumn and the proteins not containing BMAA will flow through thecolumn. The column will be washed and proteins containing BMAA will beeluted from the column as a semi-pure solution. The use of such columnsallows us to rapidly purify protein solutions such that there is agreater likelihood of obtaining proteins containing BMAA. Once isolated,the proteins will be digested using enzymes before analysis of thefragments using mass spectrometry and protein analysis software.

Example 8

This example describes proposed human clinical trials of L-Serine as atreatment for progressive neurodegenerative illness.

Based on the above considerations L-Serine and its structuralderivatives are expected to function as both a prophylactic that canprevent human progressive degenerative illness, as well as a treatmentthat will slow the progression of symptoms in patients who have beendiagnosed with progressive neurodegenerative illness.

Human clinical trials will be performed to determine if L-Serine canslow the progression of ALS and Alzheimer's, or possibly even preventtheir occurrence. To approve any compound as a drug, the U.S. Food andDrug Administration (FDA) requires three different phases of humanclinical trials. Phase I trials typically involve a small number ofpatients (10-12) with the disease, and are designed to see if the drugis safe and well-tolerated by patients. Phase II trials involve morepatients (30-60) and are designed to see if the drug is effective intreating the disease. Phase III trials are designed to reconfirm bothsafety and efficacy, and to determine possible side effects. Thesetrials typically involve 300-500 patients.

Based on the in vitro and in vivo data previously detailed, it would beunethical to present ALS patients with a placebo in a randomizeddouble-blind clinical trial. Accordingly, historical data on control ALSpatients from previous clinical trials conducted by the PhoenixNeurological Associates and others will be used.

In the current design, a Phase I open label trial of 20 ALS patientswill receive L-Serine orally at doses ranging from 500 mg/day to 30g/day for six months. ALS Inclusion Criteria: Age 18-85; Male or Female;clinically diagnosed with probable or definite ALS based on El Escorialcriteria; ALS-FRS score >25; and able to provide informed consent to andcomply with all medical procedures. ALS Exclusion Criteria: Patientswith FVC below 60%; evidence of any MND for over 3 years; any otherco-morbid condition that could make completion of trial unlikely;history of intolerance to L-serine; taking any other trial medications;and if female, pregnant or breast-feeding, or if of childbearing age,any unwillingness to prevent pregnancy until trials are completed;

Although the Phase I trial is designed to demonstrate safety of L-Serinedosing for ALS patients, within these dose ranges there could be apossible slowing of the reduction in symptoms by using the ALSFRS-R, arating of progressive Amyotrophic Lateral Sclerosis based on clinicalsymptoms. In addition, blood plasma and cerebral spinal fluid sampleswill be withdrawn and analyzed using triple quadrupole LC/MS/MS andOrbital trap LC/MS/MS to determine degree of BMAA misincorporation inneuroproteins, plasma L-Serine levels. Staining and fluorescentmicroscopy studies similar to those detailed herein will be used todetect protein aggregates and degree of misfolding of key proteinsassociated with progressive neurodegeneration. In addition,neuroproteins that are particularly vulnerable to BMAA misincorporationwill be identified using orbital trap mass spectrometry.

What is claimed:
 1. A non-human primate model of a neurodegnerativedisease, wherein the model is produced by administration of BMAA to thenon-human primate.
 2. The non-human primate model of claim 1, whereinthe non-human primate is a vervet.
 3. The non-human primate of claim 1,wherein the neurodegnerative disease comprises Alzheimer's disease. 4.The non-human primate model of claim 1, wherein neuroproteins of thenon-human primate misfold or form aggregates.
 5. A method foridentifying an agent for treatment of a neurodegnerative diseasecomprising: a) contacting the non-human primate model of claim 1 with acandidate agent; and b) determining if the candidate agent inhibits orreduces neuroprotein misfolding or neuroprotein aggregates in thenon-human primate model of claim 1, wherein an inhibition or reductionneuroprotein misfolding or neuroprotein aggregates identifies thecandidate agent as an agent for treatment of a neurodegnerative disease.6. A method for identifying an agent for prophylactic treatment of aneurodegnerative disease comprising: a) contacting a non-human primateprior to administration of BMAA with a candidate agent; b) administeringBMAA to the non-human primate; and c) determining if the candidate agentinhibits or reduces neuroprotein misfolding or neuroprotein aggregatesin the non-human primate administered, wherein an inhibition orreduction neuroprotein misfolding or neuroprotein aggregates identifiesthe candidate agent as an agent for prophylactic treatment of aneurodegnerative disease.
 7. The method of claim 5 or 6, wherein thenon-human primate is a vervet.
 8. The method of claim 5 or 6, whereinthe neurodegnerative disease comprises Alzheimer's disease.
 9. Themethod of claim 5 or 6, comprising measuring neuroprotein misfolding orneuroprotein aggregates.
 10. The method of claim 5 or 6, comprisingmeasuring BMAA incorporation into neuroproteins.